CN114805539A - Preparation method and application of porcine interferon alpha 17 mutant recombinant protein - Google Patents
Preparation method and application of porcine interferon alpha 17 mutant recombinant protein Download PDFInfo
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Abstract
The invention provides a preparation method and application of a porcine interferon alpha 17 mutant recombinant protein, wherein the 66 th site of an amino acid sequence shown in SEQ ID NO.1 is mutated from leucine (L) to phenylalanine (F), the 102 th site of the amino acid sequence is mutated from glutamic acid (E) to aspartic acid (D), and the C end of the amino acid sequence is connected with an amino acid sequence QDRLRKKE. The preparation method of the recombinant protein comprises the following steps: introducing enzyme cutting sites at both ends of the porcine interferon alpha 17 mutant and synthesizing; the synthesized fragment and pVB220 plasmid are subjected to enzyme digestion, purified and connected by T4 ligase; transforming DH5 alpha competent cells, screening positive clones, inoculating and culturing; carrying out temperature induction expression on the inclusion recombinant expression plasmid colonies; centrifuging the bacterial liquid after the culture is finished, and collecting thalli; and (3) carrying out ultrasonic crushing on the thalli, then resuspending the inclusion body, and carrying out separation and purification to obtain the microbial inoculum. The recombinant protein is applied to preparation of VSV resistant or PRV resistant drugs or vaccines.
Description
Technical Field
The invention belongs to the technical field of gene modification, and particularly relates to a preparation method and application of a porcine interferon alpha 17 mutant recombinant protein.
Background
Interferons (IFNs) are a class of cytokines that have biological functions of inhibiting tumor formation, resisting viruses, immunomodulation and regulating cell growth. According to their biological activity and antigenic activity, they are divided into three categories: the first is interferon-alpha (IFN- α) produced by virus-induced leukocytes; the second type is interferon-beta (IFN- β) produced by virus-induced fibroblasts, which shares a common surface receptor with interferon-alpha (IFN- α), and belongs to type I interferon; the third type is interferon gamma (IFN-. gamma.) produced by virus-induced lymphocytes, which is a type II interferon, unlike the surface receptors for interferon alpha (IFN-. alpha.), interferon beta (IFN-. beta.). Wherein, the I type interferon has stronger antiviral function, the II type interferon has weaker antiviral function but stronger immunoregulation function.
Porcine type I interferons primarily include IFN- α and IFN- β, with porcine IFN- α now being found in 17 subtypes and IFN- β in only 1 subtype. The homology of different porcine IFN-alpha subtype sequences is high, but the antiviral activity and other biological activities (antiviral activity, anti-inflammatory activity, MHC regulation, regulation of replicating T cells) are different due to the structural difference of different interferon subtypes. Although part of interferon products are developed at present and applied to prevention and treatment of swine infectious diseases, the clinical application of the porcine interferon still has the problems of short half-life period in vivo, high dose of frequent administration, strong systemic toxic and side effects and the like, so that the clinical application of the porcine interferon has more limitations. Therefore, the further development of a new generation of interferon with high activity, low toxicity and long acting effect for preventing and treating clinical epidemic diseases has important significance.
Disclosure of Invention
In view of the above, the present invention aims to provide a preparation method and application of porcine interferon alpha 17 mutant recombinant protein to solve the above problems. The technical scheme of the invention is as follows:
in a first aspect, the invention provides a porcine interferon alpha 17 mutant recombinant protein, wherein the 66 th site of an amino acid sequence shown in SEQ ID NO.1 is mutated from leucine (L) to phenylalanine (F), the 102 th site of the amino acid sequence is mutated from glutamic acid (E) to aspartic acid (D), and the C end of the amino acid sequence is connected with an amino acid sequence 'QDRLRKKE', the amino acid sequence of the mutant is shown in SEQ ID NO.3, and the corresponding nucleotide sequence is shown in SEQ ID NO. 4.
In a second aspect, the invention provides a preparation method of porcine interferon alpha 17 mutant recombinant protein, which comprises the following steps:
(1) introducing restriction enzyme BamH I and Hind III I enzyme cutting sites into two ends of the porcine interferon alpha 17 mutant protein, and then carrying out gene synthesis to obtain an IFN-alpha 17 gene fragment;
(2) the IFN-alpha 17 mutant gene fragment and the pVB220 plasmid are cut by restriction enzymes BamHI and Hind III and then purified, and the purified gene fragment is connected by T4 ligase;
(3) transforming a gene fragment connected by T4 ligase into DH5 alpha competent cells, screening positive clones, inoculating LB culture medium containing ampicillin for culture, carrying out enzyme digestion identification after plasmid extraction, sequencing the identified positive plasmids, and obtaining recombinant expression plasmid pVB 220-poIFN-alpha 17-7 m;
(4) inoculating the recombinant expression plasmid pVB 220-poIFN-alpha 17-7m colony to an LB culture medium containing aminobenzyl for temperature induced expression;
(5) centrifuging the bacterial solution after the culture is finished, collecting thalli, removing supernatant, adding neutral PBS (phosphate buffer solution) to wash the thalli for multiple times, suspending and mixing the thalli in a buffer solution after centrifugation, adding PMSF (permanent magnetic field) and lysozyme, and incubating for a period of time on ice;
(6) and (3) carrying out ultrasonic crushing on the thalli, adding a lysate to suspend the inclusion body, and carrying out separation and purification to obtain the microbial inoculum.
Further, the control parameters for inoculating LB culture medium containing ampicillin in the step (3) are as follows: culturing at 37 deg.C and 200rpm under shaking for 16-18 h.
Further, the control process of temperature-induced expression in step (4) is as follows: inoculating to LB culture medium containing benzyl ammonia, performing shake culture at 37 deg.C and 180rpm for 16-18h, transferring to new ampicillin positive LB culture medium, culturing at 30 deg.C and 180rpm until bacterial liquid OD is 0.6-0.8, and performing shake culture at 42 deg.C and 200rpm for 4-8 h.
Preferably, the control parameters of the centrifugal operation in the step (5) are as follows: the rotation speed is 6000rpm, and the centrifugation time is 15 min.
Further, the weight-to-volume ratio of the bacterial cells to the buffer solution in the step (5) is 1: 10.
Optionally, the buffer comprises the following components according to final concentration: NaH 2 PO 4 50mM，NaCl 300mM，pH＝8.0。
Preferably, the final concentration of PMSF in the step (5) is 1mM, and the final concentration of lysozyme is 0.2-0.4 mg/mL.
Further, in the step (6), a Ni-agarose gel purification column is used for separating and purifying the recombinant protein.
In a third aspect, the invention provides application of the porcine interferon alpha 17 mutant recombinant protein in preparation of a medicament for resisting porcine Vesicular Stomatitis Virus (VSV) or porcine pseudorabies virus (PRV).
Compared with the prior art, the invention can obtain the following technical effects:
the invention obtains a novel interferon mutant gene sequence by an amino acid mutation mode, adopts an escherichia coli expression system to express the porcine interferon alpha 17 mutant gene sequence, and adopts affinity chromatography purification to prove that the mutated porcine interferon alpha 17 protein can effectively inhibit the replication of VSV and PRV on PK-15 cells, and the antiviral activity of the porcine interferon alpha is improved compared with that of the existing porcine interferon alpha (as shown in figure 7, figure 8 and figure 9). Therefore, the porcine interferon alpha 17 mutant recombinant protein is expected to be prepared into a VSV resistant or PRV resistant medicament.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows the results of restriction enzyme analysis of recombinant expression plasmid pVB220-IFN α 17m-7m in example 1 of the present invention.
FIG. 2 is a SDS-PAGE electrophoresis of purified recombinant poIFN- α 17 and poIFN- α 17-7m proteins of example 2 of the invention.
FIG. 3 is a result chart of the identification of recombinant poIFN-. alpha.17 and poIFN-. alpha.17-7 m proteins by Western-blot experiments in example 2 of the present invention.
FIG. 4 shows the cytotoxicity of recombinant poIFN- α 17 and protein on PK-15 cells as determined in example 3 of the present invention.
FIG. 5 is a graph showing the antiviral activity of recombinant poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m proteins against VSV on PK-15 cells in example 3 of the present invention.
FIG. 6 is a graph showing the antiviral activity of recombinant poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m protein against PRV in PK-15 cells in example 3 of the present invention, in which FIG. 6-1 shows the RNA level of PRV in cells using fluorescent quantitative PCR, and FIG. 6-2 shows the detection of TCID of PRV virus in cell supernatants 50 。
FIG. 7 is a graph comparing the activity of recombinant poIFN- α 17-7m and other subtypes (poIFN- α 2, 5, 8, 14, huIFN- α 2) against VSV as detected on PK-15 cells in example 4 of the present invention.
FIG. 8 is a graph showing the comparison of anti-PRV activity of recombinant poIFN-. alpha.17-7 m and other subtypes (poIFN-. alpha.2, 5, 8, 14, huIFN-. alpha.2) detected on PK-15 cells in example 4 of the present invention, wherein FIG. 8-1 shows the PRV titer of cell supernatants, and FIG. 8-2 shows the intracellular PRV RNA level detected by fluorescent quantitative PCR.
FIG. 9 is a graph comparing the levels of recombinant poIFN-. alpha.17-7 m and other subtypes (poIFN-. alpha.2, 5, 8, 14, 17, huIFN-. alpha.2) induced interferon downstream genes (MX1, OAS1, ISG15) detected by the fluorescent quantitative PCR assay in example 3 and example 4 of the present invention.
FIG. 10 is a graph showing the in vivo antiviral activity assay of recombinant poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m proteins in example 5 of the present invention, wherein FIG. 10-1 shows lung VSV virus titers 1 to 4 days after challenge, FIG. 10-2 shows brain virus titers 1 to 4 days after challenge, FIG. 10-3 shows spinal cord virus titers 1 to 4 days after challenge, and FIG. 10-4 shows the change in mouse body weight 1 to 7 days after challenge.
Detailed Description
The porcine interferon alpha 17 mutant gene is synthesized by Shanghai bioengineering GmbH.
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1
Obtaining of porcine IFN-alpha 17 mutant and construction of porcine IFN-alpha 17 mutant recombinant plasmid
1. Experimental methods
1.1 Synthesis of the Gene of interest and construction of the recombinant plasmid
The porcine interferon alpha 17 gene sequence (poIFN-alpha 17(GenBank accession No. GQ415071)) is obtained from Genebank, as shown in SEQ ID NO:1, the sequence coding nucleotide sequence is shown in SEQ ID NO:2, after the sequence alignment and analysis are carried out by DNAstar software, a series of mutations are carried out on the amino acid site of the poIFN-alpha 17 and the carboxyl terminal sequence of the interferon sequence to find the optimal mutation mode, and the method comprises the following steps:
1) 1, mutating leucine (L) to phenylalanine (F) at position 66 in the amino acid sequence shown as SEQ ID NO.1, and naming the mutant as1 (poIFN-alpha 17-1 m);
2) 1, mutating leucine (L) at the 40 th position in an amino acid sequence shown as SEQ ID NO.1 into proline (P), and naming the mutant as 2 (poIFN-alpha 17-2 m);
3) the 88 th phenylalanine (F) in the amino acid sequence shown as SEQ ID NO.1 is mutated into serine (S) which is named as mutant 3 (poIFN-alpha 17-3 m);
4) the 102 th amino acid in the amino acid sequence shown as SEQ ID NO.1 is mutated from glutamic acid (E) to aspartic acid (D) and named as mutation 4 (poIFN-alpha 17-4 m);
5) the 127 th amino acid valine (V) in the amino acid sequence shown as SEQ ID NO.1 is mutated into alanine (A) which is named as mutation 5 (poIFN-alpha 17-5 m);
6) 1, connecting an amino acid sequence QDRLRKKE at the C end of the amino acid sequence in the amino acid sequence shown as SEQ ID NO; designated mutant 6 (poIFN-. alpha.17-6 m);
7) after the mutant 1-6 is subjected to escherichia coli expression and purification, and the in vitro antiviral activity of the interferon is measured on cells (see example 3 for details), the in vitro antiviral activity of the interferon can be remarkably improved after the 66-position amino acid, the 102-position amino acid and the C-terminal amino acid are mutated at the same time, and then the mutant is constructed and named as mutant 7 (poIFN-alpha 17-7 m).
After designing the obtained mutants, BamH I and Hind III I cleavage sites were introduced upstream and downstream of the poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m mutant coding nucleotides, respectively. The nucleotide sequences of the poIFN-alpha 17 and the poIFN-alpha 17-1-7m mutant are sent to Shanghai bioengineering company Limited for gene synthesis.
1.2 construction of poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m expression vectors
The synthetic gene fragments of the poIFN-alpha 17 and the poIFN-alpha 17-1-7m mutant and the pVB220 plasmid are respectively cut by BamH I and Hind III, purified by a gel recovery kit, the purified fragments are connected by T4 ligase to transform DH5 alpha competent cells, an LB plate with ampicillin resistance is added for overnight culture, a single clone is selected, a liquid LB culture medium with ampicillin resistance is inoculated, shaking culture is carried out at 37 ℃ and 200rpm for 16-18h, plasmids are extracted, restriction endonucleases of BamH I and Hind III are used for enzyme cutting identification, and the plasmids are named as pVB 220-poIFN-alpha 17 and pVB 220-poIFN-alpha 17-1-7 m.
2. Results of the experiment
2.1 mutations in the nucleotide and amino acid sequences of poIFN-. alpha.17, poIFN-. alpha.17-1-7 m
Obtaining a porcine interferon alpha 17 gene sequence (poIFN-alpha 17(GenBank accession No. GQ415071) from Genebank, wherein the sequence is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2. after the analysis and alignment by DNAstar software, leucine (L) at the 66 th position in the shown amino acid sequence is mutated into phenylalanine (F) which is named as mutant 1 (poIFN-alpha 17-1m), the amino acid sequence is shown as SEQ ID NO.5, leucine (L) at the 40 th position is mutated into proline (P) which is named as mutant 2 (poIFN-alpha 17-2m), the amino acid sequence is shown as SEQ ID NO.6, phenylalanine (F) at the 88 th position is mutated into serine (S) which is named as mutant 3 (poIFN-alpha 17-3m), the amino acid sequence is shown as SEQ ID NO.7, and the amino acid 102 is mutated into aspartic acid (D) from glutamic acid (E), named as mutation 4 (poIFN-alpha 17-4m), and the amino acid sequence is shown in SEQ ID NO. 8; the 127 th amino acid valine (V) is mutated into alanine (A), named as mutation 5 (poIFN-alpha 17-5m), and the amino acid sequence is shown as SEQ ID NO. 9; the C end of the amino acid sequence is connected with an amino acid sequence 'QDRLRKKE'; the amino acid sequence named as mutant 6 (poIFN-alpha 17-6m) is shown as SEQ ID NO. 10; the amino acids at position 66, 102 and C-terminal were mutated at the same time and named mutant 7 (poIFN-. alpha.17-7 m). Wherein the antiviral activity of the poIFN-alpha 17-7m is better than that of other mutants (see the embodiment 3 in detail), the amino acid sequence of the mutant 7 is shown as SEQ ID NO.3, and the corresponding nucleotide sequence is shown as SEQ ID NO. 4. The interferon poIFN-alpha 17 and (poIFN-alpha 17-1-7m) nucleotide sequences are added with BamH I and Hind III respectively at the upstream and downstream, and then sent to Shanghai bioengineering Co.
2.2 construction of the expression vectors for poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m
The constructed expression plasmid pVB 220-poIFN-alpha 17-1-7m is subjected to double enzyme digestion by BamH I and Hind III, and after agarose gel electrophoresis, target bands appear at the positions of 3700bp, 543bp and 567bp, and are consistent with the expected sizes, as shown in FIG. 1. The plasmid is sent to Shanghai bioengineering company Limited for sequencing, and the result is correct.
Example 2
Identification of recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m
1. Experimental methods
1.1 expression and purification of recombinant poIFN-alpha 17, poIFN-alpha 17-1-7m
Selecting a monoclonal from positive strains of pVB220-IFN alpha 17 and pVB220-IFN alpha 17-1-7m, inoculating an ampicillin-resistant LB liquid culture medium, carrying out shake culture at 37 ℃ and 180rpm for 16-18h, then transferring a new ampicillin-positive LB culture medium, and carrying out shake culture at 30 ℃ and 180rpm until the OD value of the bacterial liquid is between 0.6 and 0.8. Transferring the bacterial liquid to 42 ℃ and carrying out shake culture for 4-6h under the condition of 200 rpm; the cells were collected, centrifuged at 6000rpm for 15min, the supernatant was discarded, and the cells were washed with PBS (pH 7.0), centrifuged at 6000rpm for 15min, and washed once again. According to the cell ratio, buffer (50mM NaH) was dissolved 2 PO 4 300mM NaCl, pH 8.0)1:10(W/V) and the cells were suspended and mixed, and added to PMSF in a final concentration of 1mM and 0.2-0.4 mg/ml-mL lysozyme, incubated on ice for 20 min. And (3) carrying out ultrasonic treatment on the thalli by using an ultrasonic crusher, setting the power to be 5%, carrying out ultrasonic treatment for 5s and carrying out intermittent treatment for 5s, and carrying out ultrasonic treatment for 25min until bacterial liquid is clarified. The disruption solution was transferred to a centrifuge tube, and centrifuged at 15000g and 4 ℃ for 20min, and the supernatant was discarded. Using cell lysate (50mM NaH) 2 PO 4 300mM NaCl, 6M guanidenehydrochloride, pH 8.0) inclusion bodies were suspended. And (3) separating and purifying the recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m proteins by using a Ni-agarose gel purification column. Effluent liquid is collected in sections, and one tube is collected in each column volume to detect the purified protein respectively.
1.2 characterization of expressed and purified Interferon proteins by SDS-PAGE
And carrying out SDS-PAGE electrophoresis on the expressed recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m proteins, and detecting the expression and purification of the target protein.
1.3 identification of expressed and purified Interferon proteins by Western-blot
And (3) carrying out SDS-PAGE electrophoresis on the purified recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m proteins, transferring the target protein to an NC membrane, and transferring the voltage at 23V for 15 min. The antibody of the murine anti-porcine interferon is adopted to carry out a western-blot test to detect the specificity of the expressed protein.
2. Results of the experiment
2.1 expression and purification of the poIFN-alpha 17, poIFN-alpha 17-1-7m proteins
After SDS-PAGE electrophoresis is carried out on the expressed protein, the result proves that the protein with the size of 19KD is arranged at the expected position, and Ni is used for the protein with the size consistent with the expected result - After the agarose gel purification column was used for protein affinity chromatography, the results showed that the protein purity was more than 95%, and the results are shown in fig. 2.
2.2Western-blot experiment to identify the expression of poIFN-alpha 17 and poIFN-alpha 17-1-7m
The results of western-blot experiments using antibodies against porcine interferon and mouse show that the expressed recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m have good specificity, and the results are shown in FIG. 3.
Example 3
In vitro antiviral Activity assay for recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m
1. Experimental methods
1.1 recombinant poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m cytotoxicity assays
The cytotoxicity of recombinant poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m against PK-15 was examined using CellTiter96@ AQueous one solution cell promotion assay (Promega Biometrics). 24h after adding recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m to the PK-15 cell monolayer, 20. mu.L of CellTiter was added
The Aqueous single solution reagent is directly added into a culture well of a 96 cell plate and incubated for 1-4 h. The absorbance at 490nm was then recorded on a 96-well plate reader. The absorbance value measured at 490nm is directly proportional to the number of viable cells in the culture.
1.2 detection of recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m antiviral Activity
The activity of the expressed recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m proteins against VSV was first tested on PK-15 cells. PK-15 cells were plated in 96-well plates, 1 well per well ╳ 10 4 And (4) cells. After the cells grew as a monolayer, the expressed α 17 and poIFN- α 17-1-7m protein were diluted 2 fold and added to the wells, 4 replicates per protein dilution, 5% CO at 37 deg.C 2 After 24h of culture, VSV virus (1000PFU) was inoculated, recombinant poIFN-. alpha.12m was compared to poIFN-. alpha.117m protein antiviral activity, and control of uninfected and interferon-untreated cells was set. After 24h, the cells were washed with physiological saline, the cell fixing staining solution was added into the wells, treated at room temperature for 1h, the staining fixing solution was discarded, and after washing with tap water for 3-5 times, 200uL of 2-methoxyethanol was added to dissolve the staining solution. The cell culture plate was read using a spectrophotometer at 550 nm. The percentage of antiviral activity was calculated by the following formula: (OD 550) +IFN –OD550 -IFN )x 100/(OD550 Mock –OD550 -IFN ).OD550 +IFN And andOD550 -IFN OD values of interferon-treated cells and interferon-untreated cells after VSV infection and gentian violet staining, respectively; OD550 Mock OD values of cells not infected with VSV were shown.
At the same time, the PRV activity of the expressed recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m protein is detected on PK-15 cells, and the control of uninfected and interferon-untreated cells is detected. Treating PK15 cells with 1ng/mL poIFN-alpha 17m protein for 24h, discarding the liquid in the pores, inoculating PRV (PFU is 1000), collecting the cells after 24h, detecting the content of PRV virus in the cells by a fluorescence quantitative PCR method, and collecting the supernatant to detect the virus TCID 50 。
1.3 recombinant poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m induce the expression level of interferon downstream gene (ISG)
And (3) treating PK-15 cells with 1ng/mL of purified recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m protein for 24h, collecting the cells, and setting a control of uninfected and interferon-untreated cells. The induction levels of downstream genes ISG15, Mx1 and OSA1 of the interferon are detected by a fluorescent quantitative PCR method.
2. Results of the experiment
2.1 determination of PK-15 cytotoxicity of recombinant poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m
After PK-15 cells were treated with recombinant poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m, 20. mu.L of Cell Titer was added
The Aqueous single solution reagent is directly added into a culture well of a 96 cell plate and incubated for 1-4 h. Then recording the absorbance at 490nm on a 96-well plate reader, and the result shows that the absorbance of the poIFN-alpha 17 and poIFN-alpha 17-1-7m treated cells and the blank control cells has no obvious difference, thereby proving that the recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m have no obvious cytotoxicity to PK-15, and the result is shown in FIG. 4.
2.2 detection of antiviral Activity of recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m were tested on PK-15 cells using plaque reduction assay for their activity against VSV, and the results of the tests demonstrated that both recombinant poIFN-alpha 17 and poIFN-alpha 17-1-7m were effective in inhibiting VSV replication on PK-15 cells, wherein the antiviral activity of poIFN-alpha 17-7m was significantly increased over that of poIFN-alpha 17, and the minimal concentration of interferon that completely inhibits viral replication was 0.5ng/mL (FIG. 5). After PK-15 cells were treated with 1ng/mL of recombinant poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m expressed, PRV was inoculatedAfter the cells are collected, the replication level of the viral genes in the cells is detected by fluorescent quantitative PCR. At the same time, cell supernatants were collected and their TCID was determined 50 The results demonstrate that both poIFN-alpha 17-1-7m can effectively inhibit PRV replication in PK-15 cells (FIG. 6). And the antiviral activity of the poIFN-alpha 17-7m is obviously improved compared with that of the IFN-alpha 17.
2.3 recombinant poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m induce the expression level of interferon downstream gene (ISG)
The expression levels of Mx1, OAS1 and ISG15 in the cells treated by the recombinant poIFN-alpha 17 and the poIFN-alpha 17-1-7m are detected by adopting a fluorescent quantitative PCR method, and meanwhile, an interferon untreated group is set. As shown in FIG. 9, the results showed that both poIFN-. alpha.17 and poIFN-. alpha.17-1-7 m were able to effectively induce the up-regulation of the expression levels of Mx1, OAS1 and ISG15, compared to the control group, but the inhibitory effect of mutant 7 was significantly higher than that of the other interferon-treated cell groups.
Example 4
Comparison of in vitro Activity of recombinant poIFN- α 17-7m and PoIFN- α 2, - α 5, - α 8, - α 14, - α 17, and huIFN- α 2 subtypes
1. Experimental methods
1.1 comparison of recombinant poIFN- α 17-7m and poIFN- α 2, - α 5, - α 8, - α 14, - α 17 and huIFN- α 2 subtypes for in vitro antiviral Activity
First, on PK-15 cells, the activity of the recombinant poIFN-alpha 17-7m and poIFN-alpha 2, -alpha 5, -alpha 8, -alpha 14, -alpha 17, huIFN-alpha 2 proteins against VSV was compared. PK-15 cells were plated in 96-well plates, 1 well per well ╳ 10 4 And (4) cells. After the cells grew into monolayers, recombinant poIFN- α 17-7m and poIFN- α 2, - α 5, - α 8, - α 14, - α 17, huIFN- α 2 protein were diluted 2 fold and added to the wells, 4 replicates per protein dilution, 37 ℃, 5% CO 2 After 24h of culture, VSV virus (1000PFU) was inoculated and control of uninfected and interferon-untreated cells was set. After 24h, the cells were washed with physiological saline, the cell fixing staining solution was added into the wells, treated at room temperature for 1h, the staining fixing solution was discarded, and after washing with tap water for 3-5 times, 200uL of 2-methoxyethanol was added to dissolve the staining solution. The cell culture plate was read using a spectrophotometer at 550 nm. Calculating antiviral activity by formulaPercent sex, the formula is the same as in section 1.2 of example 3.
And simultaneously, on PK-15 cells, detecting the anti-PRV activity of the recombinant poIFN-alpha 17-7m and the poIFN-alpha 2, -alpha 5, -alpha 8, -alpha 14, -alpha 17 and huIFN-alpha 2 proteins, and setting a control of uninfected and interferon-untreated cells. Treating PK15 cells with 1ng/mL of poIFN-alpha 17-7m and poIFN-alpha 2, -alpha 5, -alpha 8, -alpha 14, -alpha 17 and huIFN-alpha 2 proteins for 24h, then discarding the liquid in the wells, inoculating PRV (PFU is 1000), collecting the cells after 24h, detecting the content of PRV virus in the cells by a fluorescent quantitative PCR method, and collecting the supernatant to detect the virus TCID 50.
1.2 Interferon-induced interferon downstream Gene (ISG) expression level
After PK-15 cells were treated with 1ng/mL of expressed poIFN-. alpha.17-1-7 m, poIFN-. alpha.2, -alpha.5, -alpha.8, -alpha.14, -alpha.17, huIFN-. alpha.2 protein for 24h, the cells were harvested, while a control of uninfected, interferon-untreated cells was set. And detecting the induction level of each recombinant interferon on interferon downstream genes ISG15, Mx1 and OSA1 by adopting a fluorescent quantitative PCR method.
2. Results of the experiment
2.1 detection of antiviral Activity of recombinant poIFN- α 17 and poIFN- α 2, - α 5, - α 8, - α 14, - α 17, huIFN- α 2 subtypes, as shown in FIGS. 9 and 10
The anti-VSV activity of poIFN- α 17 and poIFN- α 2, - α 5, - α 8, - α 14, - α 17, huIFN- α 2 was tested on PK-15 cells using a cellular virus inhibition assay. The detection results prove that both poIFN-alpha 17 and poIFN-alpha 2, -alpha 5, -alpha 8, -alpha 14, -alpha 17 and huIFN-alpha 2 can effectively inhibit the replication of VSV on PK-15 cells, wherein the antiviral activity of the poIFN-alpha 17-7m is obviously improved compared with that of other interferons, and the interferon concentration for completely inhibiting the viral replication is 0.5ng/mL (as shown in figure 7).
PK-15 cells are treated by using 1ng/mL of poIFN-alpha 17 and poIFN-alpha 2, -alpha 5, -alpha 8, -alpha 14, -alpha 17 and huIFN-alpha 2, and the replication level of virus genes in the cells is detected by using fluorescent quantitative PCR. At the same time, TCID was found in the cell supernatant 50 The results of the assay (as shown in FIG. 8) demonstrated that pIFN-. alpha.17 and pIFN-. alpha.2, -alpha.5, -alpha.8, -alpha.14, -alpha.17, huIFN-. alpha.2 treated PK-15 cellsThe antiviral activity of the poIFN-alpha 17-7m is obviously superior to that of other interferon groups.
2.2 Interferon-inducible interferon downstream Gene (ISG) expression level
The expression levels of Mx1, OAS1 and ISG15 in the cells treated by the poIFN-alpha 17-7m and the poIFN-alpha 2, -alpha 5, -alpha 8, -alpha 14, -alpha 17 and huIFN-alpha 2 are detected by a fluorescent quantitative PCR method, and meanwhile, an interferon untreated group is set. The results showed that the level of expression of Mx1, OAS1, ISG15 induced by poIFN- α 17-1-7m was significantly increased compared to other interferon-treated and untreated groups, as shown in FIG. 9.
Example 5
Safety and antiviral activity assay of recombinant poIFN-alpha 17m and poIFN-alpha 2, -alpha 5, -alpha 8, -alpha 14, -alpha 17, huIFN-alpha 2 subtype expression proteins in animals
1. Experimental methods
1.1 in vivo safety assay
In order to understand the safety of the proteins expressed by the PoIFN-alpha 17-7m and the PoIFN-alpha 17 subtypes in animals, 20 female mice were purchased in the experiment, and the PoIFN-alpha 17-7m and the PoIFN-alpha 17 subtypes of interferon were administered to the abdominal cavity. After the injection is finished, the mice are observed 2-3 times a day. The behavioral status and local systemic responses of the mice were observed. The safety of the product was investigated.
1.2 antiviral Activity assay
60 female mice were purchased, and the mice were divided into 4 groups, a) blank: injecting 200uLPBS buffer solution into the abdominal cavity of the mouse as a negative control; b) virus control group: injecting 200uL VSV virus liquid into the abdominal cavity; c) after 1 day of intraperitoneal injection of VSV virus, mice are given intraperitoneal injection of PoIFN-alpha 17-7m subtype interferon for treatment. All mice were weighed at-2, 0, 1, 2, 3, 4, 5, 6, 7 days post infection. After euthanasia, the brain, lung and lymph nodes of the mice were collected and the viral load of each internal organ was detected by RT-PCR.
2. Results of the experiment
2.1 in vivo safety assay
After all mice are injected with interferon, no obvious abnormal expression exists in mental state and growth, no local and general adverse symptoms occur, all mice are healthy and alive, and no death condition occurs. The results demonstrate that both recombinant poIFN-. alpha.17-7 m and poIFN-. alpha.17 are safe for mice.
2.2 antiviral Activity assay
The results of the experiment showed that the body weight of the VSV-infected mice decreased significantly on day 2 after infection, and thereafter, the body weight increased day by day, compared to the blank control group. Collecting organs to extract RNA, detecting VSV viral load of each organ by adopting a fluorescent quantitative PCR method, and proving that the VSV viral load in each organ of a poIFN-alpha 17-7m treatment group is obviously lower than that of a poIFN-alpha 17 interferon treatment group and a virus control group 1-3 days after interference injection. Meanwhile, no VSV viral RNA was detected in the blank control group. The results demonstrate that the recombinant poIFN-alpha 17-7m protein has a significantly better effect in inhibiting VSV replication in vivo than the poIFN-alpha 17 group (as shown in FIG. 10).
In conclusion, the porcine interferon alpha 17 protein after mutation can effectively inhibit the replication of VSV and PRV on PK-15 cells, and has higher antiviral activity compared with the existing porcine interferon alpha. Therefore, the porcine interferon alpha 17 mutant recombinant protein is expected to be prepared into a VSV resistant or PRV resistant medicament.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> institute of zootechnics of academy of agricultural sciences of Henan province
<120> preparation methods and applications of porcine interferon alpha 17 mutant protein and recombinant protein
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Claims (10)
1. A porcine interferon alpha 17 mutant recombinant protein is characterized in that: in the amino acid sequence shown in SEQ ID NO.1, the 66 th site is mutated from leucine (L) into phenylalanine (F), the 102 th site is mutated from glutamic acid (E) into aspartic acid (D), and the C end of the amino acid sequence is connected with an amino acid sequence 'QDRLRKKE', the amino acid sequence of the mutant is shown in SEQ ID NO.3, and the corresponding nucleotide sequence is shown in SEQ ID NO. 4.
2. A preparation method of porcine interferon alpha 17 mutant recombinant protein is characterized in that: the method comprises the following steps:
(1) introducing restriction enzyme BamH I and Hind III sites into both ends of the porcine interferon alpha 17 mutant protein of claim 1 for gene synthesis to obtain IFN-alpha 17 gene fragment;
(2) the IFN-alpha 17 mutant gene fragment and the pVB220 plasmid are purified after being cut by restriction enzymes BamH I and Hind III, and the purified gene fragment is connected by T4 ligase;
(3) transforming a gene fragment connected by T4 ligase into DH5 alpha competent cells, screening positive clones, inoculating LB culture medium containing ampicillin for culture, carrying out enzyme digestion identification after plasmid extraction, sequencing the identified positive plasmids, and obtaining recombinant expression plasmid pVB 220-poIFN-alpha 17-7 m;
(4) inoculating the recombinant expression plasmid pVB 220-poIFN-alpha 17-7m colony to an LB culture medium containing aminobenzyl for temperature induced expression;
(5) centrifuging the bacterial solution after the culture is finished, collecting thalli, removing supernatant, adding neutral PBS (phosphate buffer solution) to wash the thalli for multiple times, suspending and uniformly mixing the thalli in a buffer solution after centrifugation, adding PMSF (permanent magnetic field) and lysozyme, and incubating for a period of time on ice;
(6) and (3) carrying out ultrasonic crushing on the thalli, adding a lysate to suspend the inclusion body, and carrying out separation and purification to obtain the microbial inoculum.
3. The method for preparing porcine interferon alpha 17 mutant recombinant protein according to claim 2, characterized in that: the control parameters for inoculating the LB culture medium containing the ampicillin in the step (3) for culture are as follows: shaking and culturing at 37 deg.C and 200rpm for 16-18 h.
4. The method for preparing porcine interferon alpha 17 mutant recombinant protein according to claim 2, characterized in that: the control process of the temperature induced expression in the step (4) is as follows: inoculating to LB culture medium containing benzyl ammonia, performing shake culture at 37 deg.C and 180rpm for 16-18h, transferring to new ampicillin positive LB culture medium, culturing at 30 deg.C and 180rpm until bacterial liquid OD is 0.6-0.8, and performing shake culture at 42 deg.C and 200rpm for 4-8 h.
5. The method for preparing porcine interferon alpha 17 mutant recombinant protein according to claim 2, characterized in that: the control parameters of the centrifugal operation in the step (5) are as follows: the rotation speed is 6000rpm, and the centrifugation time is 15 min.
6. The method for preparing recombinant protein of porcine interferon alpha 17 mutant according to claim 2, wherein: the weight-volume ratio of the bacteria to the buffer solution in the step (5) is 1: 10.
7. The porcine interferon alpha 17 mutant body weight of claim 6A method for preparing a histone protein, comprising: the buffer solution comprises the following components according to final concentration: NaH 2 PO 4 50mM，NaCl 300mM，pH＝8.0。
8. The method for preparing porcine interferon alpha 17 mutant recombinant protein according to claim 2, characterized in that: in the step (5), the final concentration of PMSF is 1mM, and the final concentration of lysozyme is 0.2-0.4 mg/mL.
9. The method for preparing porcine interferon alpha 17 mutant recombinant protein according to claim 2, characterized in that: and (4) adopting a Ni-agarose gel purification column to separate and purify the recombinant protein in the step (6).
10. Use of the porcine interferon alpha 17 mutant recombinant protein according to claim 1 or the porcine interferon alpha 17 mutant recombinant protein obtained by the preparation method according to any one of claims 2 to 9 in preparation of anti-VSV or anti-PRV drugs.
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| CN110343164A (en) * | 2019-08-22 | 2019-10-18 | 安阳工学院 | A kind of pig interferon alpha-mutant and its preparation method and application of 7 site mutation of high activity |
| WO2020041636A1 (en) * | 2018-08-23 | 2020-02-27 | Exalt Therapeutics, Llc | Modified ifnl3 polypeptides comprising a pharmacokinetic enhancing moiety and their uses |
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| WO2020041636A1 (en) * | 2018-08-23 | 2020-02-27 | Exalt Therapeutics, Llc | Modified ifnl3 polypeptides comprising a pharmacokinetic enhancing moiety and their uses |
| CN110343164A (en) * | 2019-08-22 | 2019-10-18 | 安阳工学院 | A kind of pig interferon alpha-mutant and its preparation method and application of 7 site mutation of high activity |
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